Search results for: fluorescence in situ hybridization
486 Co-Precipitation Method for the Fabrication of Charge-Transfer Molecular Crystal Nanocapsules
Authors: Rabih Al-Kaysi
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When quasi-stable solutions of 9-methylanthracene (pi-electron donor, 0.0005 M) and 1,2,4,5-Tetracyanobenzene (pi-electron acceptor, 0.0005 M) in aqueous sodium dodecyl sulfate (SDS, 0.025 M) were gently mixed, uniform-shaped rectangular charge-transfer nanocrystals precipitated out. These red colored charge-transfer (CT) crystals were composed of a 1:1-mole ratio of acceptor/ donor and are highly insoluble in water/SDS solution. The rectangular crystals morphology is semi hollow with symmetrical twin pockets reminiscent of nanocapsules. For a typical crop of nanocapsules, the dimensions are 21 x 6 x 0.5 microns with an approximate hollow volume of 1.5 x 105 nm3. By varying the concentration of aqueous SDS, mixing duration and incubation temperature, we can control the size and volume of the nanocapsules. The initial number of CT seed nanoparticles, formed by mixing the D and A solutions, determined the number and dimensions of the obtained nanocapsules formed after several hours of incubation under still conditions. Prolonged mixing of the donor and acceptor solutions resulted in plenty of initial seeds hence smaller nanocapsules. Short mixing times yields less seed formation and larger micron-sized capsules. The addition of Doxorubicin in situ with the quasi-stable solutions while mixing leads to the formation of CT nanocapsules with Doxorubicin sealed inside. The Doxorubicin can be liberated from the nanocapsules by cracking them using ultrasonication. This method can be extended to other binary CT complex crystals as well.Keywords: charge-transfer, nanocapsules, nanocrystals, doxorubicin
Procedia PDF Downloads 213485 Use of Statistical Correlations for the Estimation of Shear Wave Velocity from Standard Penetration Test-N-Values: Case Study of Algiers Area
Authors: Soumia Merat, Lynda Djerbal, Ramdane Bahar, Mohammed Amin Benbouras
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Along with shear wave, many soil parameters are associated with the standard penetration test (SPT) as a dynamic in situ experiment. Both SPT-N data and geophysical data do not often exist in the same area. Statistical analysis of correlation between these parameters is an alternate method to estimate Vₛ conveniently and without additional investigations or data acquisition. Shear wave velocity is a basic engineering tool required to define dynamic properties of soils. In many instances, engineers opt for empirical correlations between shear wave velocity (Vₛ) and reliable static field test data like standard penetration test (SPT) N value, CPT (Cone Penetration Test) values, etc., to estimate shear wave velocity or dynamic soil parameters. The relation between Vs and SPT- N values of Algiers area is predicted using the collected data, and it is also compared with the previously suggested formulas of Vₛ determination by measuring Root Mean Square Error (RMSE) of each model. Algiers area is situated in high seismic zone (Zone III [RPA 2003: réglement parasismique algerien]), therefore the study is important for this region. The principal aim of this paper is to compare the field measurements of Down-hole test and the empirical models to show which one of these proposed formulas are applicable to predict and deduce shear wave velocity values.Keywords: empirical models, RMSE, shear wave velocity, standard penetration test
Procedia PDF Downloads 338484 Fabrication of Electrospun Carbon Nanofibers-Reinforced Chitosan-Based Hydrogel for Environmental Applications
Authors: Badr M. Thamer
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The use of hydrogels as adsorbents for pollutants removal from wastewater is limited due to their high swelling properties and the difficulty in recovering them after the adsorption process. To overcome these problems, a new hydrogel nanocomposite based on chitosan-g-polyacrylic acid/oxidized electrospun carbon nanofibers (CT-g-PAA/O-ECNFs) was prepared by in-situ grafting polymerization process. The prepared hydrogel nanocomposite was used as a novel effective and highly reusable adsorbent for the removal of methylene blue (MB) from polluted water with low cost. The morphology and the structure of CT-g-PAA/O-ECNFs were investigated by numerous techniques. The effect of incorporating O-ECNFs on the swelling capability of the prepared hydrogel was explored in distillated water and MB solution at normal pH. The effect of parameters including the ratio of O-ECNFs, contact time, pH, initial concentration, and temperature on the adsorption process were explored. The adsorption isotherm and kinetic were studied by numerous non-linear models. The obtained results confirmed that the incorporation of O-ECNFs into the hydrogel network improved its ability towards MB dye removal with decreasing their swelling capacity. The adsorption process depends on the pH value of the dye solution. Additionally, the adsorption and kinetic results were fitted using the Freundlich isotherm model and pseudo second order model (PSO), respectively. Moreover, the new adsorbents can be recycled for at least five cycles keeping its adsorption capacity and can be easily recovered without loss in its initial weight.Keywords: carbon nanofibers, hydrogels, nanocomposites, water treatment
Procedia PDF Downloads 147483 Microfluidic Construction of Responsive Photonic Microcapsules for Microsensors
Authors: Lingling Shui, Shuting Xie
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As alternatives to electronic devices, optically active structures from responsive nanomaterials offer great opportunity buildup smart functional sensors. Hereby, we report on droplet microfluidics enabled construction and application of photonic microcapsules (PMCs) for colorimetric temperature microsensors, enabling miniaturization for injectable local micro-area sensing and integration for large-area sensing. Monodispersed PMCs are produced by in-situ photopolymerization of hydrogel shells of cholesteric liquid crystal (CLC)-in-water-in-oil double emulsion droplets prepared using microfluidic devices, with controllable physical structures and chemical compositions. Constructed PMCs exhibit thermal responsive structural color according to the selective Bragg reflection of CLC’s periodical helical structures within the microdroplet’s spherical confinement. Constructed PMCs with tunable size and composition have been successfully applied for monitoring the living cell extracellular temperature via co-incubation with cell suspension, and for detecting human body temperature via a flexible device from assembled PMCs. These PMCs could be flexibly applied in either micro-environment or large-area surface, enabling wide applications for precision temperature monitoring biological activities (e.g. cells or organs), optoelectronic devices working conditions (e.g. temperature indicators under extreme conditions), and etc.Keywords: droplet, microfluidics, assembly, soft materials, microsensor
Procedia PDF Downloads 81482 Evaluation of Monumental Trees in Bursa City in Terms of Cultural Landscape
Authors: Murat Zencirkiran, Nilufer Seyidoglu Akdeniz, Elvan Ender Altay, Zeynep Pirselimoglu Batman
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Monumental trees make an important contribution to the cultural interaction between societies. At the same time, monument trees, which are considered as symbols of some beliefs, are living beings that are transmitted from generation to generation. Mystical, folkloric and dimensional aspects of our cultural heritage and the link between the past and present, the memorial trees of the generations of the stories conveyed the story of the legends at the same time with the aesthetic features of the objects attract attention. There are many monumental trees that witness historical processes in Bursa, which is a land of very different cultures from the Prusias (BC 232-192). Within this scope, monumental trees located within the boundaries of Bursa province and their contribution to urban culture were evaluated. Monument plane trees recorded in Bursa and its districts were determined by the Ministry of Environment and Urbanization, the Governorship of Bursa, the Provincial Directorate of Environment and Urbanism, the Directorate of Protection of Natural Assets, and these trees were examined in situ. As a result of the inspections made, the monument trees living today are classified according to their species. Within the scope of the study, it was determined that there were 1001 monumental tree species in different species within the boundaries of Bursa province. 71.83% of the recorded species were Platanus species and 11.79% were Pinus species. On the other hand, the stories about the contribution of cultural landscapes to the examples of living or now-disappearing examples of Bursa history from these monumental trees have been compiled and presented in the study.Keywords: Bursa, cultural landscape, landscape, monumental trees
Procedia PDF Downloads 426481 Understanding Chromosome Movement in Starfish Oocytes
Authors: Bryony Davies
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Many cell and tissue culture practices ignore the effects of gravity on cell biology, and little is known about how cell components may move in response to gravitational forces. Starfish oocytes provide an excellent model for interrogating the movement of cell components due to their unusually large size, ease of handling, and high transparency. Chromosomes from starfish oocytes can be visualised by microinjection of the histone-H2B-mCherry plasmid into the oocytes. The movement of the chromosomes can then be tracked by live-cell fluorescence microscopy. The results from experiments using these methods suggest that there is a replicable downward movement of centrally located chromosomes at a median velocity of 0.39 μm/min. Chromosomes nearer the nuclear boundary showed more restricted movement. Chromosome density and shape could also be altered by microinjection of restriction enzymes, primarily Alu1, before imaging. This was found to alter the speed of chromosome movement, with chromosomes from Alu1-injected nuclei showing a median downward velocity of 0.60 μm/min. Overall, these results suggest that there is a non-negligible movement of chromosomes in response to gravitational forces and that this movement can be altered by enzyme activity. Future directions based on these results could interrogate if this observed downward movement extends to other cell components and to other cell types. Additionally, it may be important to understand whether gravitational orientation and vertical positioning of cell components alter cell behaviour. The findings here may have implications for current cell culture practices, which do not replicate cell orientations or external forces experienced in vivo. It is possible that a failure to account for gravitational forces in 2D cell culture alters experimental results and the accuracy of conclusions drawn from them. Understanding possible behavioural changes in cells due to the effects of gravity would therefore be beneficial.Keywords: starfish, oocytes, live-cell imaging, microinjection, chromosome dynamics
Procedia PDF Downloads 104480 MIM and Experimental Studies of the Thermal Drift in an Ultra-High Precision Instrument for Dimensional Metrology
Authors: Kamélia Bouderbala, Hichem Nouira, Etienne Videcoq, Manuel Girault, Daniel Petit
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Thermal drifts caused by the power dissipated by the mechanical guiding systems constitute the main limit to enhance the accuracy of an ultra-high precision cylindricity measuring machine. For this reason, a high precision compact prototype has been designed to simulate the behaviour of the instrument. It ensures in situ calibration of four capacitive displacement probes by comparison with four laser interferometers. The set-up includes three heating wires for simulating the powers dissipated by the mechanical guiding systems, four additional heating wires located between each laser interferometer head and its respective holder, 19 Platinum resistance thermometers (Pt100) to observe the temperature evolution inside the set-up and four Pt100 sensors to monitor the ambient temperature. Both a Reduced Model (RM), based on the Modal Identification Method (MIM) was developed and optimized by comparison with the experimental results. Thereafter, time dependent tests were performed under several conditions to measure the temperature variation at 19 fixed positions in the system and compared to the calculated RM results. The RM results show good agreement with experiment and reproduce as well the temperature variations, revealing the importance of the RM proposed for the evaluation of the thermal behaviour of the system.Keywords: modal identification method (MIM), thermal behavior and drift, dimensional metrology, measurement
Procedia PDF Downloads 396479 Estimation of Shear Wave Velocity from Cone Penetration Test for Structured Busan Clays
Authors: Vinod K. Singh, S. G. Chung
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The degree of structuration of Busan clays at the mouth of Nakdong River mouth was highly influenced by the depositional environment, i.e., flow of the river stream, marine regression, and transgression during the sedimentation process. As a result, the geotechnical properties also varies along the depth with change in degree of structuration. Thus, the in-situ tests such as cone penetration test (CPT) could not be used to predict various geotechnical properties properly by using the conventional empirical methods. In this paper, the shear wave velocity (Vs) was measured from the field using the seismic dilatometer. The Vs was also measured in the laboratory from high quality undisturbed and remolded samples using bender element method to evaluate the degree of structuration. The degree of structuration was quantitatively defined by the modulus ratio of undisturbed to remolded soil samples which is found well correlated with the normalized void ratio (e0/eL) where eL is the void ratio at the liquid limit. It is revealed that the empirical method based on laboratory results incorporating e0/eL can predict Vs from the field more accurately. Thereafter, the CPT based empirical method was developed to estimate the shear wave velocity taking the effect of structuration in the consideration. The developed method was found to predict shear wave velocity reasonably for Busan clays.Keywords: level of structuration, normalized modulus, normalized void ratio, shear wave velocity, site characterization
Procedia PDF Downloads 235478 From Sampling to Sustainable Phosphate Recovery from Mine Waste Rock Piles
Authors: Hicham Amar, Mustapha El Ghorfi, Yassine Taha, Abdellatif Elghali, Rachid Hakkou, Mostafa Benzaazoua
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Phosphate mine waste rock (PMWR) generated during ore extraction is continuously increasing, resulting in a significant environmental footprint. The main objectives of this study consist of i) elaboration of the sampling strategy of PMWR piles, ii) a mineralogical and chemical characterization of PMWR piles, and iii) 3D block model creation to evaluate the potential valorization of the existing PMWR. Destructive drilling using reverse circulation from 13 drills was used to collect samples for chemical (X-ray fluorescence analysis) and mineralogical assays. The 3D block model was created based on the data set, including chemical data of the realized drills using Datamine RM software. The optical microscopy observations showed that the sandy phosphate from drills in the PMWR piles is characterized by the abundance of carbonate fluorapatite with the presence of calcite, dolomite, and quartz. The mean grade of composite samples was around 19.5±2.7% for P₂O₅. The mean grade of P₂O₅ exhibited an increasing tendency by depth profile from bottom to top of PMWR piles. 3D block model generated with chemical data confirmed the tendency of the mean grades’ variation and may allow a potential selective extraction according to %P₂O₅. The 3D block model of P₂O₅ grade is an efficient sampling approach that confirmed the variation of P₂O₅ grade. This integrated approach for PMWR management will be a helpful tool for decision-making to recover the residual phosphate, adopting the circular economy and sustainability in the phosphate mining industry.Keywords: 3D modelling, reverse circulation drilling, circular economy, phosphate mine waste rock, sampling
Procedia PDF Downloads 77477 Barrier Characteristics of Molecular Semiconductor-Based Organic/Inorganic Au/C₄₂H₂₈/n-InP Hybrid Junctions
Authors: Bahattin Abay
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Thin film of polycyclic aromatic hydrocarbon rubrene, C₄₂H₂₈ (5,6,11,12-tetraphenyltetracene), has been surfaced on Moderately Doped (MD) n-InP substrate as an interfacial layer by means of spin coating technique for the electronic modification of Au/MD n-InP structure. Ex situ annealing has been carried out at 150 °C for three minutes under a brisk flow of nitrogen for the better adhesion of the deposited film with the substrate surface. Room temperature electrical characterization has been performed on the C₄₂H₂₈/MD n-InP hybrid junctions by current-voltage (I-V) and capacitance-voltage (C-V) measurement in the dark. It has been seen that the C₄₂H₂₈/MD n-InP structure demonstrated extraordinary rectifying behavior. An effective barrier height (BH) as high as 0.743 eV, along with an ideality factor very close to unity (n=1.203), has been achieved for C₄₂H₂₈/n-InP organic/inorganic device. A thin C₄₂H₂₈ interfacial layer between Au and MD n-InP also reduce the reverse leakage current by almost four orders of magnitude and enhance the BH about 0.278 eV. This good performance of the device is ascribed to the passivation effect of organic interfacial layer between Au and n-InP. By using C-V measurement, in addition, the value of BH of the C₄₂H₂₈/n-InP organic/inorganic hybrid junctions have been obtained as 0.796 eV. It has been seen that both of the BH value (0.743 and 0.796 eV) for the organic/inorganic hybrid junction obtained I-V and C-V measurement, respectively are significantly larger than that of the conventional Au/n-InP structure (0.465 and 0.503 eV). It was also seen that the device had good sensitivity to the light under 100 mW/cm² illumination conditions. The obtained results indicated that modification of the interfacial potential barrier for Metal/n-InP junctions might be attained using polycyclic aromatic hydrocarbon thin interlayer C₄₂H₂₈.Keywords: I-V and C-V measurements, heterojunction, n-InP, rubrene, surface passivation
Procedia PDF Downloads 162476 Rice Serine/Threonine Kinase 1 Is Required for the Stimulation of OsNug2 GTPase Activity
Authors: Jae Bok Heo, Yun Mi Lee, Hee Rang Yun
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Several GTPases are required for ribosome biogenesis and assembly. We recently characterized rice (Oryza sativa) nuclear/nucleolar GTPase 2 (OsNug2), belonging to the YlqF/YawG family of GTPases, as playing a role in pre-60S ribosomal subunit maturation. To investigate the potential factors involved in regulating the function of OsNug2, yeast two-hybrid screens were carried out using OsNug2 as bait. Rice serine/threonine kinase 1 (OsSTK1) was identified as a potential interacting protein candidate. In vitro pull down and bimolecular fluorescence complementation assays confirmed the interaction between OsNug2 and OsSTK1, and like green fluorescent protein-tagged OsNug2, green fluorescent protein-tagged OsSTK1 was targeted to the nucleus of Arabidopsis protoplasts. OsSTK1 was not found to affect the GTP-binding activity of OsNug2; however, when recombinant OsSTK1 was included in OsNug2 assay reaction mixtures, OsSTK1 increased the GTPase activity of OsNug2. To test whether OsSTK1 phosphorylates OsNug2 in vitro, a kinase assay was performed. OsSTK1 was found to have weak autophosphorylation activity and strongly phosphorylated serine 209 of OsNug2. Yeast complementation testing resulted in a GAL::OsNug2(S209N) mutant-harboring yeast strain exhibiting a growth-defective phenotype on galactose medium at 39°C, divergent from that of a yeast strain harboring GAL::OsNug2. The intrinsic GTPase activity of mutant OsNug2(S209N) was found to be similar to that of OsNug2, was not fully enhanced upon weak binding of OsSTK1. Our findings reported here indicate that OsSTK1 functions as a positive regulator protein of OsNug2 by enhancing the GTPase activity of OsNug2, and that the phosphorylation of serine 209 of OsNug2 is essential for the complete function of OsNug2 in ribosome biogenesis.Keywords: OsSTK1, OsNug2, GTPase activity, GTP binding activity, phosphorylation
Procedia PDF Downloads 371475 Duplex Real-Time Loop-Mediated Isothermal Amplification Assay for Simultaneous Detection of Beef and Pork
Authors: Mi-Ju Kim, Hae-Yeong Kim
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Product mislabeling and adulteration have been increasing the concerns in processed meat products. Relatively inexpensive pork meat compared to meat such as beef was adulterated for economic benefit. These food fraud incidents related to pork were concerned due to economic, religious and health reasons. In this study, a rapid on-site detection method using loop-mediated isothermal amplification (LAMP) was developed for the simultaneous identification of beef and pork. Each specific LAMP primer for beef and pork was designed targeting on mitochondrial D-loop region. The LAMP assay reaction was performed at 65 ℃ for 40 min. The specificity of each primer for beef and pork was evaluated using DNAs extracted from 13 animal species including beef and pork. The sensitivity of duplex LAMP assay was examined by serial dilution of beef and pork DNAs, and reference binary mixtures. This assay was applied to processed meat products including beef and pork meat for monitoring. Each set of primers amplified only the targeted species with no cross-reactivity with animal species. The limit of detection of duplex real-time LAMP was 1 pg for each DNA of beef and pork and 1% pork in a beef-meat mixture. Commercial meat products that declared the presence of beef and/or pork meat on the label showed positive results for those species. This method was successfully applied to detect simultaneous beef and pork meats in processed meat products. The optimized duplex LAMP assay can identify simultaneously beef and pork meat within less than 40 min. A portable real-time fluorescence device used in this study is applicable for on-site detection of beef and pork in processed meat products. Thus, this developed assay was considered to be an efficient tool for monitoring meat products.Keywords: beef, duplex real-time LAMP, meat identification, pork
Procedia PDF Downloads 224474 Synthesis of Pd@ Cu Core−Shell Nanowires by Galvanic Displacement of Cu by Pd²⁺ Ions as a Modified Glassy Carbon Electrode for the Simultaneous Determination of Dihydroxybenzene Isomers Speciation
Authors: Majid Farsadrouh Rashti, Parisa Jahani, Amir Shafiee, Mehrdad Mofidi
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The dihydroxybenzene isomers, hydroquinone (HQ), catechol (CC) and resorcinol (RS) have been widely recognized as important environmental pollutants due to their toxicity and low degradability in the ecological environment. Speciation of HQ, CC and RS is very important for environmental analysis because they co-exist of these isomers in environmental samples and are too difficult to degrade as an environmental contaminant with high toxicity. There are many analytical methods have been reported for detecting these isomers, such as spectrophotometry, fluorescence, High-performance liquid chromatography (HPLC) and electrochemical methods. These methods have attractive advantages such as simple and fast response, low maintenance costs, wide linear analysis range, high efficiency, excellent selectivity and high sensitivity. A novel modified glassy carbon electrode (GCE) with Pd@ Cu/CNTs core−shell nanowires for the simultaneous determination of hydroquinone (HQ), catechol (CC) and resorcinol (RS) is described. A detailed investigation by field emission scanning electron microscopy and electrochemistry was performed in order to elucidate the preparation process and properties of the GCE/ Pd/CuNWs-CNTs. The electrochemical response characteristic of the modified GPE/LFOR toward HQ, CC and RS were investigated by cyclic voltammetry, differential pulse voltammetry (DPV) and Chronoamperometry. Under optimum conditions, the calibrations curves were linear up to 228 µM for each with detection limits of 0.4, 0.6 and 0.8 µM for HQ, CC and RS, respectively. The diffusion coefficient for the oxidation of HQ, CC and RS at the modified electrode was calculated as 6.5×10⁻⁵, 1.6 ×10⁻⁵ and 8.5 ×10⁻⁵ cm² s⁻¹, respectively. DPV was used for the simultaneous determination of HQ, CC and RS at the modified electrode and the relative standard deviations were 2.1%, 1.9% and 1.7% for HQ, CC and RS, respectively. Moreover, GCE/Pd/CuNWs-CNTs was successfully used for determination of HQ, CC and RS in real samples.Keywords: dihydroxybenzene isomers, galvanized copper nanowires, electrochemical sensor, Palladium, speciation
Procedia PDF Downloads 128473 Heterogeneity of Soil Moisture and Its Impacts on the Mountainous Watershed Hydrology in Northwest China
Authors: Chansheng He, Zhongfu Wang, Xiao Bai, Jie Tian, Xin Jin
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Heterogeneity of soil hydraulic properties directly affects hydrological processes at different scales. Understanding heterogeneity of soil hydraulic properties such as soil moisture is therefore essential for modeling watershed ecohydrological processes, particularly in hard to access, topographically complex mountainous watersheds. This study maps spatial variations of soil moisture by in situ observation network that consists of sampling points, zones, and tributaries, and monitors corresponding hydrological variables of air and soil temperatures, evapotranspiration, infiltration, and runoff in the Upper Reach of the Heihe River Watershed, a second largest inland river (terminal lake) with a drainage area of over 128,000 km² in Northwest China. Subsequently, the study uses a hydrological model, SWAT (Soil and Water Assessment Tool) to simulate the effects of heterogeneity of soil moisture on watershed hydrological processes. The spatial clustering method, Full-Order-CLK was employed to derive five soil heterogeneous zones (Configuration 97, 80, 65, 40, and 20) for soil input to SWAT. Results show the simulations by the SWAT model with the spatially clustered soil hydraulic information from the field sampling data had much better representation of the soil heterogeneity and more accurate performance than the model using the average soil property values for each soil type derived from the coarse soil datasets. Thus, incorporating detailed field sampling soil heterogeneity data greatly improves performance in hydrologic modeling.Keywords: heterogeneity, soil moisture, SWAT, up-scaling
Procedia PDF Downloads 346472 Electrochemical Sensing of L-Histidine Based on Fullerene-C60 Mediated Gold Nanocomposite
Authors: Sanjeeb Sutradhar, Archita Patnaik
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Histidine is one of the twenty-two naturally occurring essential amino acids exhibiting two conformations, L-histidine and D-histidine. D-Histidine is biologically inert, while L-histidine is bioactive because of its conversion to neurotransmitter or neuromodulator histamine in both brain as well as central nervous system. The deficiency of L-histidine causes serious diseases like Parkinson’s disease, epilepsy and the failure of normal erythropoiesis development. Gold nanocomposites are attractive materials due to their excellent biocompatibility and are easy to adsorb on the electrode surface. In the present investigation, hydrophobic fullerene-C60 was functionalized with homocysteine via nucleophilic addition reaction to make it hydrophilic and to successively make the nanocomposite with in-situ prepared gold nanoparticles with ascorbic acid as reducing agent. The electronic structure calculations of the AuNPs@Hcys-C60 nanocomposite showed a drastic reduction of HOMO-LUMO gap compared to the corresponding molecules of interest, indicating enhanced electron transportability to the electrode surface. In addition, the electrostatic potential map of the nanocomposite showed the charge was distributed over either end of the nanocomposite, evidencing faster direct electron transfer from nanocomposite to the electrode surface. This nanocomposite showed catalytic activity; the nanocomposite modified glassy carbon electrode showed a tenfold higher kₑt, the electron transfer rate constant than the bare glassy carbon electrode. Significant improvement in its sensing behavior by square wave voltammetry was noted.Keywords: fullerene-C60, gold nanocomposites, L-Histidine, square wave voltammetry
Procedia PDF Downloads 250471 Measurement of Solids Concentration in Hydrocyclone Using ERT: Validation Against CFD
Authors: Vakamalla Teja Reddy, Narasimha Mangadoddy
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Hydrocyclones are used to separate particles into different size fractions in the mineral processing, chemical and metallurgical industries. High speed video imaging, Laser Doppler Anemometry (LDA), X-ray and Gamma ray tomography are previously used to measure the two-phase flow characteristics in the cyclone. However, investigation of solids flow characteristics inside the cyclone is often impeded by the nature of the process due to slurry opaqueness and solid metal wall vessels. In this work, a dual-plane high speed Electrical resistance tomography (ERT) is used to measure hydrocyclone internal flow dynamics in situ. Experiments are carried out in 3 inch hydrocyclone for feed solid concentrations varying in the range of 0-50%. ERT data analysis through the optimized FEM mesh size and reconstruction algorithms on air-core and solid concentration tomograms is assessed. Results are presented in terms of the air-core diameter and solids volume fraction contours using Maxwell’s equation for various hydrocyclone operational parameters. It is confirmed by ERT that the air core occupied area and wall solids conductivity levels decreases with increasing the feed solids concentration. Algebraic slip mixture based multi-phase computational fluid dynamics (CFD) model is used to predict the air-core size and the solid concentrations in the hydrocyclone. Validation of air-core size and mean solid volume fractions by ERT measurements with the CFD simulations is attempted.Keywords: air-core, electrical resistance tomography, hydrocyclone, multi-phase CFD
Procedia PDF Downloads 379470 Bioprospecting for Indigenous Ruderal Plants with Potentials for Phytoremediation of Soil Heavy Metals in the Southern Guinea Savanna of North Western Nigeria
Authors: Sunday Paul Bako, Augustine Uwanekwu Ezealor, Yahuza Tanimu
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In a study to evaluate the response of indigenous ruderal plants to the metal deposition regime imposed by anthropogenic modification in the Southern Guinea Savanna of north Western Nigeria during the dry and wet seasons, herbaceous plants and samples of soils were collected in three 5m by 5m quadrats laid around the environs of the Kaduna Refinery and Petrochemical Company and the banks of River Kaduna. Heavy metal concentration (Cd, Ni, Cr, Cu, Fe, Mn and Zn) in soil and plant samples was determined using Energy Dispersive X-ray Fluorescence. Concentrations of heavy metals in soils were generally observed to be higher during the wet season in both locations although the differences were not statistically significant (P > 0.05). Concentrations of Cd, Zn, Cr, Cu and Ni in all the plants observed were found to be below levels described as phytotoxic to plants. However, above ‘normal’ concentrations of Cr was observed in most of the plant species sampled. The concentrations of Cr, Cu, Ni and Zn in soils around the KRPC and RKB were found to be above the acceptable limits. Although no hyper accumulator plant species was encountered in this study, twenty (20) plant species were identified to have high bioconcentration (BCF > 1.0) of Cd and Cu, which indicated tolerance of these plants to excessive or phytotoxic concentrations of these metals. In addition, they generally produce high above ground biomass, due to rapid vegetative growth. These are likely species for phytoextraction. Elevated concentration of metals in both soil and plant materials may cause a decrease in biodiversity due to direct toxicity. There are also risks to humans and other animals due to bioaccumulation across the food chain. There are further possibilities of further evaluating and genetically improving metal tolerance traits in some of these plant species in relation to their potential use in phytoremediation programmes in metal polluted sites.Keywords: bioprospecting, phytoremediation, heavy metals, Nigeria
Procedia PDF Downloads 284469 Synthesis and Characterization of Carboxymethyl Cellulose-Chitosan Based Composite Hydrogels for Biomedical and Non-Biomedical Applications
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Hydrogels have attracted much academic and industrial attention due to their unique properties and potential biomedical and non-biomedical applications. Limitations on extending their applications have resulted from the synthesis of hydrogels using toxic materials and complex irreproducible processing techniques. In order to promote environmental sustainability, hydrogel efficiency, and wider application, this study focused on the synthesis of composite hydrogels matrices from an edible non-toxic crosslinker-citric acid (CA) using a simple low energy processing method based on carboxymethyl cellulose (CMC) and chitosan (CSN) natural polymers. Composite hydrogels were developed by chemical crosslinking. The results demonstrated that CMC:2CSN:CA exhibited good performance properties and super-absorbency 21× its original weight. This makes it promising for biomedical applications such as chronic wound healing and regeneration, next generation skin substitute, in situ bone regeneration and cell delivery. On the other hand, CMC:CSN:CA exhibited durable well-structured internal network with minimum swelling degrees, water absorbency, excellent gel fraction, and infra-red reflectance. These properties make it a suitable composite hydrogel matrix for warming effect and controlled and efficient release of loaded materials. CMC:2CSN:CA and CMC:CSN:CA composite hydrogels developed also exhibited excellent chemical, morphological, and thermal properties.Keywords: citric acid, fumaric acid, tartaric acid, zinc nitrate hexahydrate
Procedia PDF Downloads 151468 Evaluation of DNA Oxidation and Chemical DNA Damage Using Electrochemiluminescent Enzyme/DNA Microfluidic Array
Authors: Itti Bist, Snehasis Bhakta, Di Jiang, Tia E. Keyes, Aaron Martin, Robert J. Forster, James F. Rusling
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DNA damage from metabolites of lipophilic drugs and pollutants, generated by enzymes, represents a major toxicity pathway in humans. These metabolites can react with DNA to form either 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG), which is the oxidative product of DNA or covalent DNA adducts, both of which are genotoxic and hence considered important biomarkers to detect cancer in humans. Therefore, detecting reactions of metabolites with DNA is an effective approach for the safety assessment of new chemicals and drugs. Here we describe a novel electrochemiluminescent (ECL) sensor array which can detect DNA oxidation and chemical DNA damage in a single array, facilitating a more accurate diagnostic tool for genotoxicity screening. Layer-by-layer assembly of DNA and enzyme are assembled on the pyrolytic graphite array which is housed in a microfluidic device for sequential detection of two type of the DNA damages. Multiple enzyme reactions are run on test compounds using the array, generating toxic metabolites in situ. These metabolites react with DNA in the films to cause DNA oxidation and chemical DNA damage which are detected by ECL generating osmium compound and ruthenium polymer, respectively. The method is further validated by the formation of 8-oxodG and DNA adduct using similar films of DNA/enzyme on magnetic bead biocolloid reactors, hydrolyzing the DNA, and analyzing by liquid chromatography-mass spectrometry (LC-MS). Hence, this combined DNA/enzyme array/LC-MS approach can efficiently explore metabolic genotoxic pathways for drugs and environmental chemicals.Keywords: biosensor, electrochemiluminescence, DNA damage, microfluidic array
Procedia PDF Downloads 367467 Analysis of the Engineering Judgement Influence on the Selection of Geotechnical Parameters Characteristic Values
Authors: K. Ivandic, F. Dodigovic, D. Stuhec, S. Strelec
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A characteristic value of certain geotechnical parameter results from an engineering assessment. Its selection has to be based on technical principles and standards of engineering practice. It has been shown that the results of engineering assessment of different authors for the same problem and input data are significantly dispersed. A survey was conducted in which participants had to estimate the force that causes a 10 cm displacement at the top of a axially in-situ compressed pile. Fifty experts from all over the world took part in it. The lowest estimated force value was 42% and the highest was 133% of measured force resulting from a mentioned static pile load test. These extreme values result in significantly different technical solutions to the same engineering task. In case of selecting a characteristic value of a geotechnical parameter the importance of the influence of an engineering assessment can be reduced by using statistical methods. An informative annex of Eurocode 1 prescribes the method of selecting the characteristic values of material properties. This is followed by Eurocode 7 with certain specificities linked to selecting characteristic values of geotechnical parameters. The paper shows the procedure of selecting characteristic values of a geotechnical parameter by using a statistical method with different initial conditions. The aim of the paper is to quantify an engineering assessment in the example of determining a characteristic value of a specific geotechnical parameter. It is assumed that this assessment is a random variable and that its statistical features will be determined. For this purpose, a survey research was conducted among relevant experts from the field of geotechnical engineering. Conclusively, the results of the survey and the application of statistical method were compared.Keywords: characteristic values, engineering judgement, Eurocode 7, statistical methods
Procedia PDF Downloads 296466 Efficacy of Preimplantation Genetic Screening in Women with a Spontaneous Abortion History with Eukaryotic or Aneuploidy Abortus
Authors: Jayeon Kim, Eunjung Yu, Taeki Yoon
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Most spontaneous miscarriage is believed to be a consequence of embryo aneuploidies. Transferring eukaryotic embryos selected by PGS is expected to decrease the miscarriage rate. Current PGS indications include advanced maternal age, recurrent pregnancy loss, repeated implantation failure. Recently, use of PGS for healthy women without above indications for the purpose of improving in vitro fertilization (IVF) outcomes is on the rise. However, it is still controversy about the beneficial effect of PGS in this population, especially, in women with a history of no more than 2 miscarriages or miscarriage of eukaryotic abortus. This study aimed to investigate if karyotyping result of abortus is a good indicator of preimplantation genetic screening (PGS) in subsequent IVF cycle in women with a history of spontaneous abortion. A single-center retrospective cohort study was performed. Women who had spontaneous abortion(s) (less than 3) and dilatation and evacuation, and subsequent IVF from January 2016 to November 2016 were included. Their medical information was extracted from the charts. Clinical pregnancy was defined as presence of a gestational sac with fetal heart beat detected on ultrasound in week 7. Statistical analysis was performed using SPSS software. Total 234 women were included. 121 out of 234 (51.7%) underwent karyotyping of the abortus, and 113 did not have the abortus karyotyped. Embryo biopsy was performed on 3 or 5 days after oocyte retrieval, followed by embryo transfer (ET) on a fresh or frozen cycle. The biopsied materials were subjected to microarray comparative genomic hybridization. Clinical pregnancy rate per ET was compared between PGS and non-PGS group in each study group. Patients were grouped by two criteria: karyotype of the abortus from previous miscarriage (unknown fetal karyotype (n=89, Group 1), eukaryotic abortus (n=36, Group 2) or aneuploidy abortus (n=67, Group 3)), and pursuing PGS in subsequent IVF cycle (pursuing PGS (PGS group, n=105) or not pursuing PGS (non-PGS group, n=87)). The PGS group was significantly older and had higher number of retrieved oocytes and prior miscarriages compared to non-PGS group. There were no differences in BMI and AMH level between those two groups. In PGS group, the mean number of transferable embryos (eukaryotic embryo) was 1.3 ± 0.7, 1.5 ± 0.5 and 1.4 ± 0.5, respectively (p = 0.049). In 42 cases, ET was cancelled because all embryos biopsied turned out to be abnormal. In all three groups (group 1, 2, and 3), clinical pregnancy rates were not statistically different between PGS and non-PGS group (Group 1: 48.8% vs. 52.2% (p=0.858), Group 2: 70% vs. 73.1% (p=0.730), Group 3: 42.3% vs. 46.7% (p=0.640), in PGS and non-PGS group, respectively). In both groups who had miscarriage with eukaryotic and aneuploidy abortus, the clinical pregnancy rate between IVF cycles with and without PGS was not different. When we compare miscarriage and ongoing pregnancy rate, there were no significant differences between PGS and non-PGS group in all three groups. Our results show that the routine application of PGS in women who had less than 3 miscarriages would not be beneficial, even in cases that previous miscarriage had been caused by fetal aneuploidy.Keywords: preimplantation genetic diagnosis, miscarriage, kpryotyping, in vitro fertilization
Procedia PDF Downloads 181465 Indoor Air Pollution of the Flexographic Printing Environment
Authors: Jelena S. Kiurski, Vesna S. Kecić, Snežana M. Aksentijević
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The identification and evaluation of organic and inorganic pollutants were performed in a flexographic facility in Novi Sad, Serbia. Air samples were collected and analyzed in situ, during 4-hours working time at five sampling points by the mobile gas chromatograph and ozonometer at the printing of collagen casing. Experimental results showed that the concentrations of isopropyl alcohol, acetone, total volatile organic compounds and ozone varied during the sampling times. The highest average concentrations of 94.80 ppm and 102.57 ppm were achieved at 200 minutes from starting the production for isopropyl alcohol and total volatile organic compounds, respectively. The mutual dependences between target hazardous and microclimate parameters were confirmed using a multiple linear regression model with software package STATISTICA 10. Obtained multiple coefficients of determination in the case of ozone and acetone (0.507 and 0.589) with microclimate parameters indicated a moderate correlation between the observed variables. However, a strong positive correlation was obtained for isopropyl alcohol and total volatile organic compounds (0.760 and 0.852) with microclimate parameters. Higher values of parameter F than Fcritical for all examined dependences indicated the existence of statistically significant difference between the concentration levels of target pollutants and microclimates parameters. Given that, the microclimate parameters significantly affect the emission of investigated gases and the application of eco-friendly materials in production process present a necessity.Keywords: flexographic printing, indoor air, multiple regression analysis, pollution emission
Procedia PDF Downloads 197464 Axial Load Capacity of Drilled Shafts from In-Situ Test Data at Semani Site, in Albania
Authors: Neritan Shkodrani, Klearta Rrushi, Anxhela Shaha
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Generally, the design of axial load capacity of deep foundations is based on the data provided from field tests, such as SPT (Standard Penetration Test) and CPT (Cone Penetration Test) tests. This paper reports the results of axial load capacity analysis of drilled shafts at a construction site at Semani, in Fier county, Fier prefecture in Albania. In this case, the axial load capacity analyses are based on the data of 416 SPT tests and 12 CPTU tests, which are carried out in this site construction using 12 boreholes (10 borings of a depth 30.0 m and 2 borings of a depth of 80.0m). The considered foundation widths range from 0.5m to 2.5 m and foundation embedment lengths is fixed at a value of 25m. SPT – based analytical methods from the Japanese practice of design (Building Standard Law of Japan) and CPT – based analytical Eslami and Fellenius methods are used for obtaining axial ultimate load capacity of drilled shafts. The considered drilled shaft (25m long and 0.5m - 2.5m in diameter) is analyzed for the soil conditions of each borehole. The values obtained from sets of calculations are shown in different charts. Then the reported axial load capacity values acquired from SPT and CPTU data are compared and some conclusions are found related to the mentioned methods of calculations.Keywords: deep foundations, drilled shafts, axial load capacity, ultimate load capacity, allowable load capacity, SPT test, CPTU test
Procedia PDF Downloads 104463 Non-Invasive Techniques of Analysis of Painting in Forensic Fields
Authors: Radka Sefcu, Vaclava Antuskova, Ivana Turkova
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A growing market with modern artworks of a high price leads to the creation and selling of artwork counterfeits. Material analysis is an important part of the process of assessment of authenticity. Knowledge of materials and techniques used by original authors is also necessary. The contribution presents possibilities of non-invasive methods of structural analysis in research on paintings. It was proved that unambiguous identification of many art materials is feasible without sampling. The combination of Raman spectroscopy with FTIR-external reflection enabled the identification of pigments and binders on selected artworks of prominent Czech painters from the first half of the 20th century – Josef Čapek, Emil Filla, Václav Špála and Jan Zrzavý. Raman spectroscopy confirmed the presence of a wide range of white pigments - lead white, zinc white, titanium white, barium white and also Freeman's white as a special white pigment of painting. Good results were obtained for red, blue and most of the yellow areas. Identification of green pigments was often impossible due to strong fluorescence. Oil was confirmed as a binding medium on most of the analyzed artworks via FTIR - external reflection. Collected data present the valuable background for the determination of art materials characteristic for each painter (his palette) and its development over time. Obtained results will further serve as comparative material for the authentication of artworks. This work has been financially supported by the project of the Ministry of the Interior of the Czech Republic: The Development of a Strategic Cluster for Effective Instrumental Technological Methods of Forensic Authentication of Modern Artworks (VJ01010004).Keywords: non-invasive analysis, Raman spectroscopy, FTIR-external reflection, forgeries
Procedia PDF Downloads 172462 Characterization, Antibacterial and Cytotoxicity Evaluation of Silver Nanoparticles Synthesised Using Grewia lasiocarpa E. Mey. Ex Harv. Plant Extracts
Authors: Nneka Augustina Akwu, Yougasphree Naidoo
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Molecular advancement in technology has created a means whereby the atoms and molecules (solid forms) of certain materials such as plants, can now be reduced to a range of 1-100 nanometres. Green synthesis of silver nanoparticles (AgNPs) was carried out at room temperature (RT) 25 ± 2°C and 80°C, using the metabolites in the aqueous extracts of the leaves and stem bark of Grewia lasiocarpa as reductants and stabilizing agents. The biosynthesized AgNPs were characterized by UV-Vis spectrophotometry, attenuated total reflectance - Fourier transforms infrared (ATR-FTIR) spectroscopy, nanoparticle tracking analysis (NTA), Energy Dispersive X-ray fluorescence scanning electron microscope (SEM-EDXRF) and high-resolution transmission electron microscopy (HRTEM). The AgNPs were biologically evaluated for antioxidant, antibacterial and cytotoxicity activities. The phytochemical and FTIR analyses revealed the presence of metabolites that act as reducing and capping agents, while the UV-Vis spectroscopy of the biosynthesized NPs showed absorption between 380-460 nm, confirming AgNP synthesis. The Zeta potential values were between -9.1 and -20.6 mV with a hydrodynamics diameter ranging from 38.3 to 46.7 nm. SEM and HRTEM analyses revealed that AgNPs were predominately spherical with an average particle size of 2- 31 nm for the leaves and 5-27 nm for the stem bark. The cytotoxicity IC50 values of the AgNPs against HeLa, Caco-2 and MCF-7 were >1 mg/mL. The AgNPs were sensitive to all strains of bacteria used, with methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) being more sensitive to the AgNPs. Our findings propose that antibacterial and anticancer agents could be derived from these AgNPs of G. lasiocarpa, and warrant their further investigation.Keywords: antioxidant, cytotoxicity, Grewia lasiocarpa, silver nanoparticles, Zeta potentials
Procedia PDF Downloads 141461 Development of Environmentally Clean Construction Materials Using Industrial Waste from Kazakhstan
Authors: Galiya Zhanzakovna Alzhanova, Yelaman Kanatovich Aibuldinov, Zhanar Baktybaevna Iskakova, Gaziz Galymovich Abdiyussupov, Madi Toktasynuly Omirzak, Aizhan Doldashevna Gazizova
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The sustainable use of industrial waste has recently increased due to increased environmental problems in landfills. One of the best ways to utilise waste is as a road base material. Industrial waste is a less costly and more efficient way to strengthen local soils than by introducing new additive materials. This study explored the feasibility of utilising red mud, blast furnace slag, and lime production waste to develop environmentally friendly construction materials for stabilising natural loam. Four different ratios of red mud (20, 30, and 40%), blast furnace slag (25, 30, and 35%), lime production waste (4, 6, and 8%), and varied amounts of natural loam were combined to produce nine different mixtures. The results showed that the sample with 40% red mud, 35% blast furnace slag, and 8% lime production waste had the highest strength. The sample's measured compressive strength for 90 days was 7.38 MPa, its water resistance for the same period was 7.12 MPa, and its frost resistance for the same period was 7.35 MP; low linear expansion met the requirements of the Kazakh regulations for first-class building materials. The study of mineral composition showed that there was no contamination with heavy metals or dangerous substances. Road base materials made of red mud, blast furnace slag, lime production waste, and natural loam mix can be employed because of their durability and environmental performance. The chemical and mineral composition of raw materials was determined using X-ray diffraction, X-ray fluorescence, scanning electron microscopy, energy dispersive spectroscopy, atomic absorption spectroscopy, and axial compressive strength were examined.Keywords: blast furnace slag, lime production waste, natural loam stabilizing, red mud, road base material
Procedia PDF Downloads 110460 Low-Complex, High-Fidelity Two-Grades Cyclo-Olefin Copolymer (COC) Based Thermal Bonding Technique for Sealing a Thermoplastic Microfluidic Biosensor
Authors: Jorge Prada, Christina Cordes, Carsten Harms, Walter Lang
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The development of microfluidic-based biosensors over the last years has shown an increasing employ of thermoplastic polymers as constitutive material. Their low-cost production, high replication fidelity, biocompatibility and optical-mechanical properties are sought after for the implementation of disposable albeit functional lab-on-chip solutions. Among the range of thermoplastic materials on use, the Cyclo-Olefin Copolymer (COC) stands out due to its optical transparency, which makes it a frequent choice as manufacturing material for fluorescence-based biosensors. Moreover, several processing techniques to complete a closed COC microfluidic biosensor have been discussed in the literature. The reported techniques differ however in their implementation, and therefore potentially add more or less complexity when using it in a mass production process. This work introduces and reports results on the application of a purely thermal bonding process between COC substrates, which were produced by the hot-embossing process, and COC foils containing screen-printed circuits. The proposed procedure takes advantage of the transition temperature difference between two COC grades foils to accomplish the sealing of the microfluidic channels. Patterned heat injection to the COC foil through the COC substrate is applied, resulting in consistent channel geometry uniformity. Measurements on bond strength and bursting pressure are shown, suggesting that this purely thermal bonding process potentially renders a technique which can be easily adapted into the thermoplastic microfluidic chip production workflow, while enables a low-cost as well as high-quality COC biosensor manufacturing process.Keywords: biosensor, cyclo-olefin copolymer, hot embossing, thermal bonding, thermoplastics
Procedia PDF Downloads 239459 Controlled Growth of Charge Transfer Complex Nanowire by Physical Vapor Deposition Method Using Dielectrophoretic Force
Authors: Rabaya Basori, Arup K. Raychaudhuri
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In recent years, a variety of semiconductor nanowires (NWs) has been synthesized and used as basic building blocks for the development of electronic and optoelectronic nanodevices. Dielectrophoresis (DEP) has been widely investigated as a scalable technique to trap and manipulate polarizable objects. This includes biological cells, nanoparticles, DNA molecules, organic or inorganic NWs and proteins using electric field gradients. In this article, we have used DEP force to localize nanowire growth by physical vapor deposition (PVD) method as well as control of NW diameter on field assisted growth of the NWs of CuTCNQ (Cu-tetracyanoquinodimethane); a metal-organic charge transfer complex material which is well known of resistive switching. We report a versatile analysis platform, based on a set of nanogap electrodes, for the controlled growth of nanowire. Non-uniform electric field and dielectrophoretic force is created in between two metal electrodes, patterned by electron beam lithography process. Suspended CuTCNQ nanowires have been grown laterally between two electrodes in the vicinity of electric field and dielectric force by applying external bias. Growth and diameter dependence of the nanowires on external bias has been investigated in the framework of these two forces by COMSOL Multiphysics simulation. This report will help successful in-situ nanodevice fabrication with constrained number of NW and diameter without any post treatment.Keywords: nanowire, dielectrophoretic force, confined growth, controlled diameter, comsol multiphysics simulation
Procedia PDF Downloads 192458 Precious Gold and Diamond Accessories Versus False Fashion Diamond and Stained Accessories
Authors: Amira Yousef Mahrous Yousef
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This paper includes fast fashion verses sustainable fashion or slow fashion Indian based consumers. The expression ‘Fast fashion’ is generally referred to low-cost clothing collections that considered first hand copy of luxury brands, sometime interchangeably used with ‘mass fashion’. Whereas slow fashion or limited fashion which are consider to be more organic or eco-friendly. "Sustainable fashion is ethical fashion and here the consumer is just not design conscious but also social-environment conscious". Paper will deal with desire of young Indian consumer towards such luxury brands present in India, and their understanding of sustainable fashion, how to maintain the equilibrium between never newer fashion, style, and fashion sustainability. The green fashion market is growing rapidly as eco-friendly consumers are willing to expand their organic lifestyle to include clothing. With an increasing share of fashion consumers globally, Indian consumers are observed to consider the social and environmental ethics while making purchasing decisions. While some research clearly identifies the efforts of responsible consumers towards green fashion, some argue that fashion-orientated consumers who are sensitive towards environment do not actively participate towards supporting green fashion. This study aims to analyze the current perception of green fashion among Indian consumers. A small-scale exploratory study is conducted where consumers’ perception of green fashion is examined followed by an analysis of translation of this perception into purchase decision making. This research paper gives insight into consumer awareness on green fashion and provides scope towards the expansion of ethical fashion consumption .Keywords: inclusions, temperature gradient, HPHT synthetic fibers, polyamide fibers, fiber volume, compressive strength. gold nano clusters, copper ions, wool keratin, fluorescence
Procedia PDF Downloads 48457 Optimization for Guide RNA and CRISPR/Cas9 System Nanoparticle Mediated Delivery into Plant Cell for Genome Editing
Authors: Andrey V. Khromov, Antonida V. Makhotenko, Ekaterina A. Snigir, Svetlana S. Makarova, Natalia O. Kalinina, Valentin V. Makarov, Mikhail E. Taliansky
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Due to its simplicity, CRISPR/Cas9 has become widely used and capable of inducing mutations in the genes of organisms of various kingdoms. The aim of this work was to develop applications for the efficient modification of DNA coding sequences of phytoene desaturase (PDS), coilin and vacuolar invertase (Solanum tuberosum) genes, and to develop a new nanoparticles carrier efficient technology to deliver the CRISPR/Cas9 system for editing the plant genome. For each of the genes - coilin, PDS and vacuolar invertase, five single RNA guide (sgRNAs) were synthesized. To determine the most suitable nanoplatform, two types of NP platforms were used: magnetic NPs (MNPS) and gold NPs (AuNPs). To test the penetration efficiency, they were functionalized with fluorescent agents - BSA * FITS and GFP, as well as labeled Cy3 small-sized RNA. To measure the efficiency, a fluorescence and confocal microscopy were used. It was shown that the best of these options were AuNP - both in the case of proteins and in the case of RNA. The next step was to check the possibility of delivering components of the CRISPR/Cas9 system to plant cells for editing target genes. AuNPs were functionalized with a ribonucleoprotein complex consisting of Cas9 and corresponding to target genes sgRNAs, and they were biolistically bombarded to axillary buds and apical meristems of potato plants. After the treatment by the best NP carrier, potato meristems were grown to adult plants. DNA isolated from this plants was sent to a preliminary fragment of the analysis to screen out the non-transformed samples, and then to the NGS. The present work was carried out with the financial support from the Russian Science Foundation (grant No. 16-16-04019).Keywords: biobombardment, coilin, CRISPR/Cas9, nanoparticles, NPs, PDS, sgRNA, vacuolar invertase
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